1. Field of the Invention
The present invention relates to the use of sulfolane in combination with an aqueous alkaline solution as a solubilization and naturation method for somatotropins.
2. Description of the Prior Art
Many methods for protein solubilization and naturation have been studied. In U.S. Pat. No. 4,511,503, refractile bodies are reported as insoluble granules of aggregated denatured somatotropin in the cytoplasm of the whole microorganism, E. Coli, in which they are produced. These refractile bodies are formed by overproduction of the specified protein of interest, such as somatotropin, as a result of genetically engineering the E. Coli cell to purposely overproduce that desired protein. The only mechanism by which to treat the refractile bodies is with a strong denaturant or chaotropic agent in order to cause the improperly folded molecules of both the desired protein product and the E. Coli proteins to unfold and become soluble. In addition to causing this denaturation, proteins must then be renatured in the proper monomeric form in order to be biologically active. This monomeric form is especially important for somatotropin. In order to obtain the active protein product, strong denaturants such as guanidine hydrochloride or urea at very high concentrations have been used.
As disclosed in U.S. Pat. No. 4,677,196 other chaotropic agents such as sodium dodecylsulfate (SDS) have also been used. Weak denaturants such as urea in concentrations of 3 to 5 molar have been used and are disclosed in U.S. Pat. No. 4,731,440. In U.S. Pat. No. 5,064,943 the deletion of urea is disclosed.
Each of the methods have certain associated problems with them. Guanidine hydrochloride is expensive and must be replaced during the naturation process in order for naturation to occur. Sodium dodecylsulfate is an effective denaturant and less expensive than guanidine hydrochloride, but it binds to the denatured protein so tightly that it makes complete removal from the protein difficult and increases processing costs. Urea is usually used as a weaker denaturant or chaotropic agent, but even methods utilizing urea have associated problems, such as contamination of the final product, handling, storage and waste treatment. Therefore, any method which uses no denaturant at all or low concentrations of a denaturant is specifically needed in the art. Methods for using no denaturant and for using low concentration of urea are disclosed in copending applications Ser. No. 07/285,477 filed Dec. 16, 1988, now U.S. Pat. No. 5,064,943 and Ser. No. 07/446,280 filed Dec. 5, 1989 respectively.
Additionally, it is the obtaining of this monomer product that is important in arriving at product proteins which have the physiological and biological activities of somatotropins. It is known that somatotropin monomer consists of approximately 191 amino acid residues and has a molecular weight of roughly 22,000 daltons. The monomer is not linked or noncovalently bonded to other similar monomer molecules.
In addition to the monomer form, somatotropins can exist in dimer form which means that two monomer molecules are covalently linked through intermolecular disulfide bonds or are noncovalently associated with one another. The dimer protein molecule consists of double the number of amino acids and double the molecular weight of monomer and is formed, unfortunately, by the inefficiencies of processes used to solubilize and renature the refractile bodies. In other words, it is formed by the isolation, naturation and purification process required to obtain active product protein from inactive inclusion (refractile) bodies combined within the microorganism cell.
The present invention provides a commercially feasible process for solubilizing proteins by effectively combining the use of sulfolane as a solubilizing agent with optimal naturation conditions used in aqueous dissolution procedures. Naturation includes oxidation of the cystine residues to form cysteine bonds. The use of sulfolane results in yield advantages when compared to the use of no denaturant, very low concentrations of urea or compared to other urea processes.